Cystic fibrosis (CF) is an inherited lung disease characterized by mucus hypersecretion, reduced mucociliary clearance of inhaled bacteria, particularly Pseudomonas aeruginosa (PA), and chronic airway inflammation. While the exact molecular mechanisms leading to lung dysfunction in CF patients remain to be clarified, PA binding to respiratory epithelial cells in the initial stages of infection and later becoming trapped within mucus plugs are important factors in the pathophysiology of the disease. Thus, further characterization of the molecular relationship between airway epithelial cells, mucus production, and PA will lead to a better understanding of CF and the generation of new treatment therapies. Respiratory mucosal epithelial cells express a variety of cell surface receptors that signal the presence of potentially harmful inhaled substances. For example, TLRs with specificity for bacterial peptidoglycan (TLR2), LPS (TLR4), and flagellin (TLR5) activate signaling pathways leading to the production of proinflammatory cytokines and chemokines and the recruitment of immune effecter cells leading to clearance of the invading microbe. Our published studies demonstrated that MUC1 mucin (MUC1 in humans, Muc1 in animals) is another pattern recognition receptor expressed on the surface of epithelial cells that binds to flagellin. We wondered why would mucosal epithelia have evolved two different receptors (TLR5, MUC1) with binding specificity for the same ligand (flagellin)? To address this question, we performed preliminary studies that led us to hypothesize that whereas TLR5 initiates inflammatory responses, MUC1 counter-regulates airway inflammation after immune clearance of the pathogen, thereby returning the lungs to homeostasis. We will test this hypothesis using the following Aims. Aim 1: To determine whether or not MUC1/Muc1 down-regulates general TLR inflammatory responses. TLRs are highly conserved proteins that signal through a common pathway leading to NF-?B activation and inflammatory cytokine/chemokine production. Therefore, Aim 1 will test the hypothesis that the anti-inflammatory effects of MUC1 are not restricted to TLR5, but are manifested with most, if not all, TLR responses. Lung inflammation in Muc1 and Muc1-/- mice will be compared following treatment with agonists for TLR2, 3, 4, 7, 8, and 9. Additionally, human airway epithelial cell lines will be treated with a MUC1 siRNA or control RNA and TLR-driven inflammatory responses will be compared. Aim 2: To compare inflammatory responses in CF airway cells expressing or non-expressing MUC1/Muc1. This Aim will test the hypothesis that airway hyperinflammation in CF is due, in part, to diminished activity of MUC1. We will compare airway inflammation in Muc1??/- single knockout and Muc1-/-/Cftr-/- double knockout mice using an experimental model of PA lung infection. For in vitro studies, CF airway epithelial cells will be transfected with the MUC1 siRNA or control RNA and TLR agonist-stimulated inflammatory responses will be determined. Successful completion of the proposed experiments will provide a foundation to apply for a major grant(s) to study the detailed mechanism of action of MUC1 in the pathogenesis of bacterial-induced hyperinflammatory lung diseases such as CF. Mucosal inflammation is an essential host defense mechanism mounted in response to pathogens. However, the mechanisms through which inflammation is resolved are poorly understood. Our preliminary results indicate that expression of MUC1 mucin by mucosal epithelial cells down-regulates ongoing inflammation during bacterial lung infection. In this proposal, we will characterize at the molecular level the anti- inflammatory mechanism of MUC1. Successful accomplishment of this project will provide pharmacological strategies to control excessive inflammation during airway bacterial colonization of mucosal surfaces.